Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA

ABSTRACT: RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate several functions at once and likely impact the structural integrity of the large mRNP assemblies, these proteins often function in. Thus, the function of the RNA-binding activity of RBPs is often unknown. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric (MS) analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components. For functional analysis, we chose Npl3, for which we identified crosslinks in its two RNA recognition motifs (RRMs) as well as in the flexible linker connecting the two. Both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in each of these three regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutant strongly impairs recruitment of some mRNP components to chromatin and the incorporation of other mRNP components into nuclear mRNPs, establishing a function of Npl3 in mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of a nuclear mRNP component, an approach that can be applied to many RBPs in any RNA metabolic process.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Alexander Wulf  

LAB HEAD: Henning Urlaub

PROVIDER: PXD035153 | Pride | 2023-03-11

REPOSITORIES: Pride